Substitution of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator.
Regulatory protein p4 of Bacillus subtilis phage phi 29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase alpha subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical alpha subunit containing the N-terminal domain of the E. coli alpha subunit and the CTD of the B. subtilis alpha subunit. Reconstitution of RNA polymerases containing this chimerical alpha subunit, the E. coli beta and beta' subunits, and the vegetative sigma factor from either E. coli (sigma 70) or B. subtilis (sigma A), generated hybrid enzymes that were responsive to protein p4 and efficiently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis alpha-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.
Mencía
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Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Monsalve
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Rojo
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Salas
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D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000821:virology; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases